The present invention relates in general to supported fluid bilayers and methods of confining them to selected areas. More specifically, the invention relates to microfabricated arrays of independently-addressable supported fluid bilayer membranes, their uses, and methods for their manufacture.
Not applicable.
Over the last several years, a number of high-throughput screening methods have been developed to facilitate the screening of thousands, if not millions, of compounds for a desired activity or activities. Such methods are typically based on detecting the binding of a potentially effective compound to a receptor. While these binding assays are effective at constraining the universe of compounds which may have the desired activity, they are typically not well-suited for evaluating this activity with any degree of detail.
The biological activity of potentially active compounds is typically evaluated using less efficient but more informative xe2x80x9csecondary screensxe2x80x9d or assays which typically require a substantial input of time by a trained technician or scientist. For evaluation of candidate compounds affecting integral membrane proteins such as receptors and ion channels, the amount of time required per compound may be several hours or days if the assay includes effects on electrophysiological activity. Accordingly, there is a need for a more efficient xe2x80x9csecondary screenxe2x80x9d of compounds affecting the activity of such integral membrane proteins and other membrane or membrane-associated components, to identify those few compounds that justify further detailed analysis.
In one aspect, the present invention includes a surface detector array device. The device includes a substrate having a surface defining a plurality of distinct bilayer-compatible surface regions separated by one or more bilayer barrier regions, a bulk aqueous phase covering the substrate surface, a lipid bilayer expanse carried on each of the bilayer-compatible surface regions, and an aqueous film interposed between each bilayer-compatible surface region and corresponding lipid bilayer expanse. In a general preferred embodiment, the bilayer-compatible surface regions and the bilayer barrier surface regions are formed of different materials.
The bilayer-compatible surface region may be formed from any of a variety of materials having such bilayer-compatible surface properties, including SiO2, MgF2, CaF2, and mica, as well as a polymer film, such as a polyacrylamide or dextran film. SiO2 is a particularly effective material for the formation of a bilayer-compatible surface region.
The bilayer barrier surface region may be formed from any of a variety of materials having such bilayer barrier surface properties, including gold, positive photoresist, aluminum oxide, and indium tin oxide.
In a general embodiment, the lipid bilayer expanse contains at least one lipid selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, and sphingomyelin.
In one embodiment, the device contains between about 10 and about 100 distinct bilayer-compatible surface regions. In another embodiment, the device contains at least about 2500 distinct bilayer-compatible surface regions. In yet another embodiment, the device contains at least about 25,000 distinct bilayer-compatible surface regions. In still another embodiment, the device contains at least about 2.5 million distinct bilayer-compatible surface regions.
The bilayer-compatible surface regions are separated from one another, in one general, embodiment, by bilayer barrier regions that are between about 1 xcexcm and about 10 xcexcm in width.
The lipid bilayer expanses on different bilayer-compatible surface regions may have different compositions, and may further include a selected biomolecule, with different expanses having a different biomolecule, such as a transmembrane receptor or ion channel. The biomolecule may be covalently or non-covalently attached to a lipid molecule. Examples of non-covalent interactions include electrostatic and specific molecular interactions, such as biotin/streptavidin interactions. Examples of biomolecules include proteins, such as ligands and receptors, as well as polynucleotides and other organic compounds.
In another aspect, the invention includes a method of forming a surface detector device having a plurality of independently-addressable lipid bilayer regions. The method includes the steps of (i) treating a planar substrate to form a substrate surface defining a plurality of distinct bilayer-compatible surface regions separated by one or more bilayer barrier regions, and (ii) applying a suspension of lipid bilayer vesicles to the plurality of distinct bilayer-compatible surface regions under conditions favorable to the formation of supported bilayers on the bilayer-compatible surface regions. The applying of the vesicles results in the formation of supported bilayer membranes on the bilayer-compatible surface regions.
In another aspect, the invention includes a method of forming a surface detector array device. The method includes the steps of (i) providing a substrate having a surface defining a plurality of distinct bilayer-compatible surface regions separated by one or more bilayer barrier regions, (ii) applying a first suspension of lipid bilayer vesicles having a first composition to a first of said plurality of distinct bilayer-compatible surface regions, (iii) applying a second suspension of lipid bilayer vesicles having a second composition to a second of said plurality of distinct bilayer-compatible surface regions, (iv) incubating the substrate with the first and second suspensions to form a first lipid bilayer expanse stably localized above the first distinct bilayer-compatible surface region and a second lipid bilayer expanse stably localized above the second distinct bilayer compatible surface region, and (v) establishing a bulk aqueous phase above the lipid bilayer expanses.
In a preferred embodiment, the applying steps of the above-described method comprise generating drops of the first and/or second suspensions on an end of a transfer device such as, e.g., a hollow ceramic tip, a hollow metal tip, a micropipette, a pin, or the like, and touching the drops to the first and second bilayer-compatible region(s). In certain embodiments, the drops comprise less than 100 nl, less than 75 nl, less than 50 nl, less than 25 nl, less than 15 nl, less than 10 nl, or less than 5 nl.
In another preferred embodiment, the applying steps of the above-described method comprise ejecting aliquots of the first and/or second suspensions from an end of a transfer device such as, e.g., a hollow ceramic tip, hollow metal tip, micropipette, an electro-piezo print head, an ink-jet print head or the like, across an air space separating the end of the transfer device from the bilayer-compatible surface regions, and onto the bilayer compatible surface region(s). In certain embodiments, the ejected aliquots comprise less than 100 nl, less than 75 nl, less than 50 nl, less than 25 nl, less than 15 nl, less than 10 nl, or less than 5 nl.
In yet another aspect, the invention includes a method for detecting a selected ligand in a mixture of ligands. The method includes the steps of (i) contacting the mixture with a biosensor surface detector array device such as described above, and (ii) detecting binding of the selected ligand to receptors which specifically bind it.
In another aspect, the invention includes a method for assaying the interaction between a test agent and a composition. The method includes the steps of (i) providing a biosensor surface detector array device such as described above, wherein said device comprises a first lipid bilayer expanse having a first composition and a second lipid bilayer expanse having a second composition different from said first composition, (ii) contacting the device with a bulk aqueous phase comprising a test agent, and (iii) assaying an interaction between the test agent and the first composition and between the test agent and the second composition.
In preferred embodiments, the lipid bilayer expanses each comprise less than 5 xcexcg of material, or less than 1 xcexcg of material, or less than 0.5 xcexcg of material.
In other preferred embodiments, the first composition comprises a first biomolecule and the second composition comprises a second biomolecule. In another preferred embodiment, the first and second biomolecules are different members of a receptor protein family. In yet another preferred embodiment, the first and second compositions comprise different lipids.
In still another aspect, the invention includes a surface detection array device for use in a biosensor. Such a device includes (i) a substrate having a surface defining a plurality of distinct bilayer-compatible surface regions separated by one or more bilayer barrier regions, (ii) a bulk aqueous phase covering the substrate surface, (iii) a lipid bilayer expanse carried on each of the bilayer-compatible surface regions, and (iv) an aqueous film interposed between each bilayer-compatible surface region and corresponding lipid bilayer expanse. Each bilayer expanse contains a specie of receptor, or biomolecule, and different bilayer expanses contain different species of receptors or biomolecules.
Another aspect of the present invention provides for a surface detector array device, comprising a substrate having a surface defining a plurality of distinct bilayer-compatible surface regions separated by one or more bilayer barrier regions, a bulk aqueous phase covering said substrate surface, a lipid bilayer expanse carried on each of said bilayer-compatible surface regions, and an aqueous film interposed between each bilayer-compatible surface region and corresponding lipid bilayer expanse, wherein said bilayer-compatible surface regions and said bilayer barrier surface regions are formed of different materials, and wherein each bilayer-expanse carried on each bilayer-compatible region is compositionally different than adjacent bilayer-expanses. Other embodiments of the invention further include a plurality of groups of said bilayer-compatible regions, wherein said groups each define an area where said bilayer-expanses are compositionally similar, and where the bilayer-expanses within different groups are compositionally different.
The invention further provides a method for forming an array of biosensor regions, where each region has a different, known lipid bilayer compositions comprising the steps of:
providing a biosensor array having a plurality of lipid bilayer compatible regions, each compatible region being surrounded by one or more bilayer barrier regions,
providing a gradient forming device loaded with two or more different lipid bilayer compositions, the gradient forming device in fluid communication with a spot forming device for forming spots on a surface,
providing a multi-axis translation table for holding and translating a biosensor array workpiece,
placing a biosensor array workpiece that has a plurality of bilayer compatible regions surrounded by one or more barrier regions, and
forming spots of mixed lipid bilayer compositions resulting from the gradient forming device forming a gradient and translating the table in at least one axis while dispensing such composition mixture as it is formed thereby dispensing to different, consecutive locations different ratios of each lipid bilayer compositions.
The invention further provides a method for making gradient biosensor array comprising the steps of: mixing together first and second different lipid bilayer forming compositions contained from first and second sources by flowing in a substantially laminar flow, two different compositions from two different sources into one mixing chamber that substantially retains the laminar flow character of the two different compositions while flowing through the mixing chamber, where the facing edges of each different composition mix to form a gradient having a first edge and a second edge and further comprising composition combinations of different ratios beginning from the first edge of the gradient that faces the first composition, and ending at the second edge of the gradient that faces the other, second composition, and where the mixing chamber is adapted to dispense the gradient in a substantially laminar flow across the surface of the array, and where the compositions contained in the gradient are captured and retained upon initial contact by bilayer-compatible regions of the array.